DE2262449B2 - Divinylfither maleic anhydride copolymers - Google Patents

Description

The invention relates to a solid cyclic copolymer of divinyl ether and maleic anhydride in the form a composition with a certain molecular weight range and molecular assemblies that are derived from divinyl ether and maleic anhydride in a ratio of 1: 2, as well as a method for its Manufacturing.

Copolymers of divinyl ether and maleic anhydride are known and in US Pat. No. 3,224,943 as Malignant tumors have a growth-inhibiting effect. In US-PS 36 24 218 these copolymers are also described as a means of combating foot and mouth disease virus. Although the im The copolymers described in the prior art are known to be effective agents against tumors and viruses, they are not yet commercially available as pharmaceuticals, which leads to a number of undesirable side effects is due. These undesirable side effects primarily include acute toxicity, the sensitization of endotoxins, the induction of anemia and a suppression of the metabolism.

It is accordingly the object of the present invention to provide an improved solid cyclic copolymer made of divinyl ether and maleic anhydride available that the aforementioned Does not have disadvantages and is nevertheless used effectively for combating tumors and fighting viruses can be.

To achieve this object, the invention proposes a solid cyclic copolymer of the type mentioned, which is characterized by a number average molecular weight of 5000 to 30,000 and an MJM _n ratio of 1.0 to 3.0.

To prepare these copolymers, it is proposed according to the invention that, for fractionation between polymers with lower and higher molecular weights, these are treated selectively, in such a way that the number of molecular assemblies in the copolymer molecule is selected so that a product with a number average molecular weight is selected of 5000-30,000 and an MJM _n ratio of 1.0-3.0.

The principle of the invention is based on the fact that the properties of the polymers are those mentioned at the outset Kind can be significantly improved if one has a material with a narrow molecular weight range and with a narrow molecular weight distribution selects. All of the polymers known from the prior art are essentially Mixtures with a wide range of molecular weights, ranging from liquid or semi-solid Copolymers with a very low molecular weight up to solid copolymers with a quite a high molecular weight extends. In

Owing to this broad range of molecular weights, individual copolymer molecules with a molecular weight of only a few hundred can occur in the copolymer mixtures, and molecules with a molecular weight of a few hundred thousand can occur at the other end of the scale. The ratio M _w to M _n can be up to 7 or more.

The copolymers according to the invention have the same or better effectiveness for the treatment of the solid EHRLICH tumor and the encephalomyocarditis virus than the copolymers according to the prior art, but compared to these are above all much less acutely toxic and sensitize endotoxins to a far lower degree , hardly cause anemia, have lower serum levels of glutamine pyruvate transaminase and suppress aminopyrene metabolism to a far lesser extent. In addition to these significant improvements, the copolymers according to the invention also lead to a lower loss of body weight, cause organ enlargement only to a significantly reduced extent, in particular with regard to the liver, lungs and spleen, and suppress phagocytosis only to a much lesser extent Measure from. The number average molecular weight of the copolymers according to the invention is preferably in the range from 10,000-25,000. The MJM _n ratio is preferably between 1.5 and 2.5. Furthermore, the molecular weight distribution range should preferably become narrower as the average molecular weight increases. The copolymers according to the invention are water-soluble after hydrolysis.

The number average molecular weights mentioned in the context of the present description are determined by membrane osmometry at 30 ^° C. in a high-speed membrane osmometer using tetrahydrofuran as the solvent and a deacetylated acetyl cellulose as the membrane.

The MJM _n ratio is a measure of the molecular weight distribution. In this ratio, M _{w is} the weight average molecular weight and M _{n is} the number average molecular weight. Both are determined by gel chromatography with the methyl ester of the polymers. To determine the MJM _n ratio, the methyl ester samples are dissolved in tetrahydrofuran and injected through a ΙΟ-μπι filter into a gel diffusion chromatograph equipped with a bank of four styrogel columns with different pore diameters. The process for operating this chromatograph and the evaluation of the results obtained have been described in J. Cazes, H. Chem. Ed., 43, A 576, A 625 (1966). More detailed information on the MJM _n ratio can be found in Billmeyer, Textbook of Polymer Science, p. 7, Interscience Pubiishers, Inc. (1966).

As is known, divinyl ether-maleic anhydride copolymers can be used can be obtained by mixing divinyl ether with maleic anhydride in an aromatic Diluent in a molar ratio of about

1: 2 copolymerized, using an initiator for the Radical mechanism [e.g. benzoyl peroxide, azo-bis (isobutyronitrile) or the like] is used.

Under these conditions, the polymer separates directly from the solvent during formation. The copolymers obtained in this way should be cyclical and can be classified as follows: reproduce.

-CH CH

II
cc

O CH

CH ₂ CH CH ₂

CH CH O O

C C

In fact, however, these copolymers obtained according to the prior art are always extensive Mixture of individual copolymer molecules with molecular weights which are extremely low up to very high values. After none of the known in the prior art method for Copolymerizing divinyl ether with maleic anhydride has hitherto made it possible to produce uniform copolymers with a narrow range of molecular weight distribution.

The homogeneous and uniform copolymers according to the invention obviously have those above specified cyclic structure, however, must in order to actually comply with the molecular weight specifications of the invention and to have the narrow molecular weight distribution range required according to the invention, are manufactured using special processes. There are also a number of procedures in Question. For example, the copolymers prepared according to the prior art can be fractionated, why, for example, on a sand column fractionation, gel permeation chromatography, ultrafiltration, fractional precipitation, or the like Procedures can be used. Alternatively, the copolymers can also be produced by the action of heat or be specifically degraded by the action of chemical reagents. Furthermore, the Copolymers according to the invention are obtained if the polymerization of the divinyl ether with the Maleic anhydride performs in solution under direct control.

In order to explain the invention in more detail, exemplary embodiments are described below.

example 1

Divinyl ether and maleic anhydride are copolymerized by a known method. The copolymer mixture thus obtained is then separated into the desired fractions by sand column fractionation. For this purpose, a polymerization vessel is charged with 10.3 parts of maleic anhydride, 200 parts of dry benzene and 7.45 parts of carbon tetrachloride. After the maleic anhydride has dissolved, the solution is flushed with nitrogen and 3.7 parts of distilled divinyl ether in 60 parts of benzene are added. The reaction vessel is heated to 8O ⁰ C. A solution of 0.073 parts of benzoyl peroxide in 5.6 parts of benzene is added with stirring. The reaction is carried out at a temperature of 80-90 ° C. for 4 hours. After the reaction has ended, the reaction slurry obtained is cooled ^{to 25-3O 0 C.} The swollen copolymer is separated off and extracted repeatedly with a mixture of 7.5 parts of benzene and 10 parts of hexane, filtered and dried in vacuo. The product obtained is a broad mixture of cyclic divinyl ether-maleic anhydride copolymers with a number average molecular weight of about 35,600 and a ratio M _w IM _n of 7.64.
The copolymer mixture thus obtained is fractionated on a sand column using the general procedures as described by John Elliot in Polymer Fractionation, MJR Cantow Editor, Chapter B 2, pages 67-93, Academic Press, New York, NY (1967) became.

In detail, 28.7 parts of the copolymer mixture obtained in this way were used in a mixture of 460 Parts of methyl ethyl ketone and 105 parts of methyl isobutyl ketone dissolved. The solution thus obtained was on a Jacketed sand column given at room temperature. A further 60 parts of the methyl ethyl ketone-methyl isobutyl ketone solvent were then added added as eluent. The column was then heated to 69 ° C. and held at this temperature for 1 hour. To rinse of the solvent mixture, benzene was then added to the column. After the column has cooled down The copolymer was fractionated with a tetrahydrofuran-n-hexane solvent mixture at room temperature. The proportions of tetrahydrofuran and η-hexane in the solvent mixture were during of the process varied, the starting mixture consisting of 70% tetrahydrofuran and 30% η-hexane and then the tetrahydrofuran proportions were gradually increased during the fractionation in such a way that that towards the end of the fractionation pure tetrahydroso furan was used. In this way, 8 different fractions of about 21 each received. Every these fractions were concentrated and the copolymer was precipitated by adding dry benzene. the Data of the copolymer fractions obtained in this way are summarized below:

fraction Number average molecular 1.66 weight*) 1.84 (Mn) 2.25 a 16 700 2.16 b 29 400 . 2.35 C. 36 200 d 45 300 e 71300

continuation

fraction

Number average molecular weight *)

(M ₁₁ )

M _n JM ₁₁ "

f 95 500 2.59 G 117,000 3.21 H 203,000 4.21

! 0

*) Determined by membrane osmometry. **) Gel penetration determined by chromatography.

Copolymer M _n M _n .
~ M ~ n Percentage
tumor
under
depression Starting
mix before
Fractionation 35 600 7.64 50 a 16 700 1.66 61 b 29 400 1.84 45 C. 36 200 2.25 45 d 45 300 2.16 67 f 95 500 2.59 32 H 203,000 4.21 75

Seed mixture examined. In addition there were 10 males each white Swiss mice a single intravenous injection of the copolymer in isotonic saline solution, as described in Example 2, administered. After 24 h the test animals were given an intravenous Injection of about 50 times the LD-50 of the encephalomyocarditis virus. The percentage Mortality of the test animals was 13 days after the Virus infection determined. The results summarized below were obtained:

15th

Example 2

Copolymer fractions obtained according to Example 1 and samples of the non-uniform starting copolymer mixture are examined with regard to their activity in suppressing tumors. For these investigations, 10 male white Swiss mice were intravenously injected with the copolymers in isotonic saline solution (2.5 mg / ml) which had been adjusted to a pH of 7.2. After 24 hours, the test animals were given a second injection. Each of the injections contained 25 mg of copolymer per kg of body weight. Approximately 48 hours after the second injection of the copolymer, each of the test animals was injected subcutaneously with an amount of 10 ⁷ solid EHRLICH adenocarcinoma cells in saline. The tumors were removed and weighed after 10 days. The percentage of tumor suppression is compared by comparing the mean tumor weight of the tumors removed from the mice treated with the copolymer with the mean weight of those tumors that were obtained from a group of 10 control mice that, apart from the treatment with the copolymer, were exactly the same as the other group of Test animals had been treated were removed. The following results were obtained:

20th

25th

30th

Copolymer M _n M _n .
M _n Percentage
Mortal
speed Starting
mix before
Fractionation 35 600 7.64 10 control - - 90 a 16 700 1.66 20th b 29 400 1.84 30th C. 36 200 2.25 10 d 45 300 2.16 10 e 71300 2.35 20th f 95 500 2.59 20th G 117,000 3.21 20th

From the above list it can be seen that both all copolymer fractions as the initial copolymer mixture are also antiviral.

As the data compiled above show, all the fractions are just like that Starting copolymer mixture effective tumor inhibitors.

Example 3

It is the antiviral activity of the fractions prepared according to Example 1 and the starting copolymer

60

Example 4

In this example the influence of samples from the various fractions prepared in Example 1 is shown and the starting copolymer mixture on the total body weight and on the weight of various Body organs examined. A Group of 10 male white Swiss mice together with 10 mice as a control experimental group used. To the experiments for the liver enlargement, spleen enlargement, for the lung and Groups of 5 male white Swiss mice each with 10 of these animals were used as thymus weights Control group used. The copolymer was administered in a single intravenous injection, as in the example 2, applied in an amount of 25 mg of copolymer per kg of body weight. To determine the The total body weight became the mean weight of each group one week after the copolymer treatment compared with the mean weight of this group before treatment. In the experiments in Within the framework of the liver enlargement, the enlargement of the spleen, within the framework of the lung weight and the thymus weight The mean weights of the organs within each group were compared with the mean total body weight compared within the group. As part of these experiments, the following Get the compiled results:

7th M _n M _n - 22nd 62 449 + 9.5 liver 8th Lungs Thymus ~ M ~ " -18.1 weight MiIz- weight
in % weight Copolymer Change of body body weight Hl / 0
body body total weight weight body weight weight _ _ + 8.2 6.1 weight 0.76 0.24 35 600 7.64 G + 0.9 7.1 0.34 1.29 0.15 control + 2.1 -11.8 1.35 Output copoly -4.0 -2.3 merisat before the 16 700 1.66 -7.7 6.5 0.89 0.22 Fractionation 29 400 1.84 6.5 0.63 1.15 0.20 a 36 200 2.25 + 1.8 6.6 0.71 1.31 0.16 b 71300 2.35 + 0.2 8.0 0.59 1.22 0.18 C. 117,000 3.21 -2.6 8.0 1.46 1.21 0.16 e -0.5 1.15 G -1.7

From the table it can be seen that the copolymers with the higher molecular weights just like the initial copolymer mixtures, a significant decrease in total body weight and the weight of the thymus and a significant increase in the weight of the liver, spleen and lung weight cause. In comparison with this, the copolymers according to the invention only cause less Change in total body weight and organ weights.

Example 5 ^J ° copolymer

In this example, samples of the copolymer fractions prepared according to Example 1 and samples of the Starting copolymer mixture examined for its acute toxicity. Standard test procedures are used for this for the determination of the LD-50 carried out by intravenous injection with white male Swiss mice. In this context, “LD-50” usually denotes the one that was lethal for 50% of the animals examined Dose. The results are in the following summary in mg of copolymer per kg Body weight compiled:

(25 mg per kg of body weight) administered in a saline solution in the manner described in Example 2. as Control group, 25 male white Swiss mice were treated in the same way, except for them a pure saline solution was injected instead of the copolymer solution. After a week the test animals Blood drawn. The amount of hemoglobin in g / 100 ml of blood was determined. The results are given below in g%:

M _n

M _n - ~ M _n

Hemoglobin level

(Gram%)

Copolymer

M _n

₁ M _n

LD-50 (mg / kg)

Output copoly 35 600 7.64 74 merisat before the Fractionation a 16 700 1.66 131 C. 36 200 2.25 100 e 71300 2.35 87

45

50

control - - 16.8 Output copoly 35 600 7.64 12.8 merisat before the Fractionation a 16 700 1.66 16.3 b 29 400 1.84 15.3 C. 36 200 2.25 14.8 e 71 300 2.35 12.2 G 117,000 3.21 11.7

It can be seen from the values shown that the copolymers with the higher molecular weights just like the starting copolymer mixtures are more toxic than the copolymers according to Invention are.

Example 6

In this example, samples of the fractions that were prepared according to Example 1 and a sample of the Starting copolymer mixture examined for its anemia-causing properties. In addition each 5 male white Swiss mice receive a single intravenous injection of the copolymer

65 From the above compilation of data it can be seen that all copolymers with a molecular weight above the molecular weight of the copolymers according to the invention, including the starting copolymer mixtures, cause a significant decrease in the hemoglobin level.

Example 7

In this example the influence of the various copolymers on the liver functions is examined. It two series of tests were carried out.

In the first series of tests, 24 hours after the intravenous injection of the copolymer (25 mg Copolymer per kg body weight) the serum level of glutamine pyruvate transaminase is determined. In there were 5 male white Swiss mice in each group and 8 of these animals in each control group examined. The determination was made according to the method described in Sigma Technical Bulletin No. 505 (Revised March 1971), Sigma Chemical Co., St. Louis, Missouri, performed the colorimetric determination procedures described. The results are given below in Sigma-Frankel-Ein

030109/166

as described in the Sigma Technical Bulletin. The growth of these units over 100 indicates liver necrosis.

In the second series of experiments, the activity of the liver microsome enzyme 24 h after the intravenous Injection of the copolymer (25 mg of copolymer per kg of body weight) determined. 5 male white each Swiss mice formed the experimental animal group and the control animal group. 24 h after the copolymerization

The liver was removed from the animal's jection and centrifuged at 9000 g. The supernatant liquid was collected. This liquid contains the enzymes that convert aminopyrene into formaldehyde can. A decrease in these enzymes indicates liver damage. The results of this second Test series are given in the form of the percentage suppression of aminopyrene metabolism.

Copolymer

M _n

M «

M _n

Sigma Frankel
units Percentage
oppression
of aminopyrene
metabolism 32 0 200 64 52 0 87 7.1 93 29.5 *) 51.8 96 56.5 205 53

control

Starting copolymer
before fractionation

a
b
c
d

e
G
*) Not determined.

35 600

16 700
29 400

36 200
45 300
71 300

117,000

7.64

1.66 1.84 2.25 2.16 2.35 3.21

As taken from the data in the table above jo

can be, practice all copolymers with molecular copolymers weight over the molecular weights of the copolymers according to the invention damaging influences on the liver.

Example 8

In this example, the effects of samples f of the fractions prepared according to Example 1 and the non-uniform starting copolymer mixture 40 ″ on the triggering sensitization of endotoxins are examined kg body weight). 24 hours later, 4 ^r> subgroups were infected by 5 mice each with different concentrations of Salmonella typhosa endotoxin. the LD 50 was determined. the results of the Endotoxinsensibilisierung are described below in the form of the LD 50 in mg Salmonella typhosa endotoxin per kg ^r > o body weight.

M _n M _n -
W _n Endotoxin
sensiti
ization,
LD-50,
mg / kg 71 300 2.35 0.48 95 500 2.59 0.48 117,000 3.21 0.48 203,000 4.21 0.48

Copolymer

M ₁ ,

M _n

Endotoxin sensiti
ization,
LD-50,
mg / kg

Control - 34

Starting copoly- 35 600 - 0.38

merisat before the

Fractionation

a 16 700 1.66 15

b 29 400 1.84 3.0

c 36 200 2.25 0.38

d 45 300 2.16 0.48

The table above clearly shows that endotoxin sensitization, which is noticeable by a decrease in the LD-50, which is clear for the copolymers with molecular weights over 30,000 increases.

Example 9

This example describes the influence of the molecular weight and the molecular weight distribution of copolymer samples on various biological activities. 8 different copolymers with the ν, molecular weights and M ", / M _n ratios listed below were investigated:

Copolymer

A molecular weight below the range according to the invention, M ₁ JM _n in the invention

area

B Molecular weight below the range according to the invention, MJM _n outside the range according to the invention
C molecular weight and MJM _n in the range according to the invention ■

D molecular weight and MJM _n within the range according to the invention

E molecular weight in the range according to the invention, MJM _n outside the range according to the invention

F molecular weight above the range according to the invention and MJM _n within the range according to the invention

G molecular weight above the range according to the invention, MJM _n outside the range according to the invention

The samples were made as follows:

Copolymer

A Direct solution polymerization
B direct copolymerization according to US Pat. No. 3,224,943
C Direct solution polymerization: a polymerization vessel was charged with 14.8 parts of maleic anhydride and 160 parts of tetrahydrofuran. The resulting solution was flushed with nitrogen, and 5.3 parts of divinyl ether in 17.7 parts of tetrahydrofuran were added. The reaction solution was cooled to 15 ° C and irradiated under nitrogen with a sun lamp (UV and visible range) for 90 minutes, the temperature being maintained in the range of 15-20 ⁰ C. Then 132 parts of η-hexane were added to precipitate the copolymer. The copolymer was purified by centrifugation and decanting, repeated washing with a mixture of 3.3 parts of benzene and 1 part of η-hexane, filtered and dried in a stream of nitrogen.
D Sand column fractionation as described in Example 1

E direct copolymerization as described in US Pat. No. 3,224,943

F sand column fractionation

K) G direct copolymerization according to the process described in US Pat. No. 3,224,943

Each of the samples became acute in terms of their influences on antitumor activity, antiviral activity Toxicity, sensitization of endotoxins, Sigma-Frankel units, Hemoglobin levels, enlarged liver and enlarged spleen after those in the previous ones Methods described in the examples examined. In addition, the white blood cells were counted.

In this test, 5 mice in each group and 25 mice in the experimental group were examined. One week after a single intravenous injection of the copolymer, the blood samples were drawn and the number of white blood cells per mm ^{3 was} determined. The results are summarized in the table below.

Copoly M _n M ₁₁ . Tumor- Antivirals Acute endo- Sigma Hemo white liver spleen merisat M ₁ , under- Activity, Toxic toxin- Frankel globin blood weight weight ITM ff press Per ity sensitive A- mirrors cells in% in% kung centual LD-50 ization units Per body body Mortal LD-50 mm ³ weight weight speed mg / kg mg / kg G% A. 3,500 2.62 41 60 *) 15th 34 *) *) *) *) B. 1500 11.0 65 60 126 15th 32 16.6 7 520 5.4 0.52 C. 6,800 2.34 67 20th 100 15th 33 *) *) *) *) D. 16 700 1.66 61 20th 131 15th 52 16.3 7,300 6.5 0.63 E. 16 800 4.45 67 10 96 0.68 120 13.2 11450 7.0 1.14 F. 71300 2.35 55 20th 87 0.48 96 12.2 10 600 8.0 1.46 G 35 600 7.6 62 10 74 0.38 190 12.3 13 500 8.4 1.27 Con _ _ 0 93 - 34 29 16.8 6 466 4.8 0.37 troll *) Not certainly.

The table above clearly shows the superiority of the copolymers according to the invention.

Claims (3)

Patent claims: 1. Solid copolymer of divinyl ether and maleic anhydride in the form of a composition with a certain molecular weight range and molecular assemblies which are derived from divinyl ether and maleic anhydride in a ratio of 1: 2, characterized by a number average molecular weight of 5000-30,000 and a ratio MJM _n of 1 , 0 - 3.0. 2. Copolymer according to claim 1, characterized by a number average molecular weight of 10,000-25,000. 3. Copolymer according to one of claims 1 or 2. characterized by a ratio MJM _n of 1.5-2.5.